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10, NO. 4, 1 9 9 0

Drug-Induced Allergic Hepatit is LANCE R. POHL, Pharm.D., Ph.D.

tase. Since these possibilities, however, have not yet been supported by human studies, the most currently accepted alternate mechanism for idiosyncratic drug-induced hepatotoxicity is an allergic or hypersensitivity reaction.?

GENERAL FEATURES OF DRUG-INDUCED ALLERGIC HEPATITIS

The diagnosis of a drug-induced allergic hepatitis, like all drug allergic reactions, relies on several riter ria.^ Drug-induced allergic hepatitis usually occurs after repeated exposures and often appears to be dose independent. It may be manifested by blood and tissue eosinophilia and fever. If the patient is rechallenged with the drug, the same pathologic condition should be observed. In most cases, the symptoms subside promptly when treatment with the drug is discontinued. The most convincing evidence that a drug-induced hepatitis has an allergic basis is the demonstration of specific antibodies or sensitized T lymphocytes in the patient's blood that react with the drug, its metabolites, normal liver tissue (autoantigen), or liver tissue that has been modified (neoantigen) by the drug or its metabolites (Table 1). In most cases, however, these tests are not done or are found to be negative, possibly because the wrong test antigen is used in the assay.' Finally, the best proof that a druginduced hepatitis has an allergic basis is the demonstration of an immunopathologic mechanism of toxicity in animals. Yet this type of proof has not been demonstrated for any drug-induced hepatitis that is believed to have an immunopathologic basis. This is important to do because drug-induced antibodies or sensitized T lymphocytes do not have to cause tissue damage"' and can arise following tissue injury by nonimmunologic mechanisms. "' It is generally assumed that for most drugs to induce an immune response they must covalently alter an endogenous macromolecule and produce a drug-carrier conjugate14 (Fig. 1). This structural modification is most likely produced by a reactive metabolite, since most drugs are chemically inert. The reactive metabolite may directly alter the macromolecule by covalently attaching to it. Alternatively, the reactive metabolite may indiFrom the Luhorcrtory 01' Chrmic.ul Phnrtncrc.olog~. Nntiot~c~l rectly alter the macromolecule by causing an alteration Hrtrrt. Lung, rrtld Blood Institute, B~the.sdcr,Mut$otld. in the splicing of the gene of the macromolecule, a soReprint requests: Dr. Pohl, Laboratory of Chemical Pharmamatic mutation of the gene of the macromolecule, a cology, National Heart, Lung, and Blood Institute. Building 10, Room 8N 115, Bethesda. MD 20892 biosynthetic post-translational modification of the Copyright 0 1990 by Thieme Medical Publishers. Inc., 381 Park Avenue South, New York, NY 10016. All r ~ g h t sreserved.

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It has been estimated that more than 600 drugs may cause hepatic injury.' The type of lesion caused by these drugs may include hepatocellular necrosis, steatosis, cholestasis, granuloma, chronic hepatitis, and cirrhosis. I 3 Drugs that cause hepatotoxicity have been classified into two categories; those that are intrinsic hepatotoxins and those that are idiosyncratic hepatotoxins.' The hepatotoxicity caused by intrinsic hepatotoxins, such as chloroform and acetaminophen, is generally host independent, dose dependent, and reproducible in animals. The majority of drugs that cause hepatotoxicity, however, appear to be idiosyncratic hepatotoxins. Their reactions are host dependent, often apparently dose independent, and difficult to reproduce in animals. They are also hard to predict and usually are not recognized as a potential medical problem until the drug has been in general use for some time. Drug-induced hepatotoxicities, whether intrinsic or idiosyncratic, are believed to be caused in many cases by reactive metabolite^.'.^ In the case of intrinsically hepatotoxic agents, reactive metabolites appear to cause toxicity either directly by covalently altering cellular macromolecules or indirectly by oxidizing cellular components through the intermediacy of reactive endogenous oxygen species, such as hydrogen peroxide, hydroxyl radical, or lipid peroxides that are products of the reactions of reactive metabolites of drugs."." This may lead to the inactivation of enzymes, to the disruption of intracellular calcium homeostasis, or to a general loss of cellular membrane integrity by lipid peroxidation.' ' Idiosyncratic drug-induced hepatic damage might also be attributed to similar processes. If this were the case, then there would have to be some reason why the toxicities are host dependent. It could be due to pharmacokinetic factors. For example, the individual might have abnormally high levels of enzymes that activate the drug into a reactive metabolite or possess a mutant form of the enzyme that is catalytically more active than the normal enzyme. Alternatively, an individual may have an abnormally low level or activity of enzymes that detoxify reactive metabolites, such as glutathione transferases, glutathione peroxidase, catalase, or superoxide dismu-

SEMINARS IN LIVER DISEASE-VOLUME

TABLE 1. Drug-induced Hepatic Disease where Immunity Against Liver Tissue or Drug Has Been Detected Immunitv Aaainst Drug

Autoantigen

Neoantigen

Drug

Reference

Iproniazid Isaxonine Dihydralazine Methyldopa Phenytoin Erythromycin Floxacillin Cloxacillin Quinidine Procainamide Nifedipine Ampicillin p-~minosalicylate Isoniazid Hydrochlorothiazide Streptomycin Reserpine Co-trimoxazole Tienilic acid Ethanol Halothane

10, NUMBER 4, 1990

toantigenic determinants, which are ordinarily seen as self. As a consequence of hapten binding or other covalent modifications of the macromolecule, however, tolerance is b y p a s ~ e d . ~ , ' ~ Probably the major reason why very little progress has been made in the understanding of how drugs produce allergic hepatitis is that until very recently little was known of the identity of hapten-carrier conjugates associated with these diseases. In this review, I will address the studies dealing with the characterization of the antigens and presumably immunogens associated with the hepatitis produced by tienilic acid, ethanol, and halothane. Only these three drugs will be discussed because very little is presently known about the antigens associated with the hepatotoxicity produced by other drugs.

TlENlLlC ACID

It has been estimated that approximately 1 in 800 patients will develop a drug-induced hepatic injury after receiving tienilic acid (ticrynafen), an antihypertensive uricosuric diuretic.16 This toxicity led to the withdrawal of the drug from sale in the United States in 1980. By indirect immunofluorescence analysis of tissue slices, *A: represents antibody immunity; C: cellular immunity. the sera of patients diagnosed with tienilic-induced hepTDiscussed in detail in the text. atitis were found to contain a specific class of autoantibodies that reacted with an undefined constituent of liver and kidney microsomes (LKM). These antibodies were macromolecule, or an oxidative modification of the maclassified as anti-LKM2 antibodies because their pattern cromolecule leading to oxidized residues or the formaof immunofluorescence staining of tissue sections diftion of abnormal disulfide linkages. In any event, once fered from the anti-LKM1 antibodies that were assoa macromolecule has been covalently altered, it may act ciated with patients diagnosed with a subtype of as an immunogen and elicit a specific humoral (antiautoimmune chronic active hevatitis. Subseauent imbody) response, a specific cellular (T lymphocyte) remunoblotting and immunoprecipitation studies with husponse, or both responses.'4 The immune response could man liver microsomes and purified proteins have shown be directed against three general classes of antigenic dethat the anti-LKM2 antibodies are directed against a 52 terminants (epitopes) of the altered m a c r ~ m o l e c u l e ' ~ kd class of cytochromes P450 known as citochrome (Fig. 1). First, the antigenic determinants can include the P450 8.'*3" This isoform of cytochrome P-450 is closely bound derivative of the drug (haptenic epitopes), or at related, if not identical, to cytochrome P-450MP. The least portions of it. Second, they can represent novel eplatter name was given to the enzyme because it hydroxitopes of the carrier molecule (also known as new antiylates the anticonvulsant drug mephenytoin (MP) in the genic determinants [NAD]) that result from its covalent 4 position of its phenyl ring.I9 In addition, it was found modification. Third, they can be native epitopes or authat the anti-LKM2 antibodies inhibited the metabolism

+ Carrier Protein

A

-

Reactive Metabolite

rU

I 1. Anti-hapten 2. Anti-NAD 3. Anti-Autoantigen

t NAD Immune Response

I

FIG. 1. Classes of immune responses that can be produced against drugcarrier conjugates. When a reactive metabolite of a drug covalently binds to an endogenous carrier macromolecule, immune responses may be induced against the covalently bound metabolite or hapten, new antigenic determinants (NAD), or autodeterminants.

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of mephenytoin19 and the 5-hydroxylation of tienilic acid" by human liver microsomes. The antibodies appear to be directed against conformational epitopes, at least in part, because several patients' antibodies only recognized the protein when it was transferred to nitrocellulose from nondenaturing gels. " More recently, some o f t h e patients diagnosed with tienilic acid hepatitis were found to have antibodies in their sera that were directed against tienilic acid-altered liver cell neoantigens, in addition to those directed against auto determinant^.^' This was shown in an antibody-dependent cell-mediated cytotoxicity (ADCC) assay in which hepatocytes from rabbits treated with tienilic acid were incubated with serum from patients diagnosed with tienilic acid hepatitis and lymphocytes from a normal patient. It was found that sera from 16 of 36 patients with presumed tienilic acid hepatotoxicity induced significant cytotoxicity to the rabbit hepatocytes. In addition, three of ten sera from patients who received tienilic acid, but did not show signs of liver damage, also induced significant ADDC to these hepatocytes. None of the sera from 20 normal controls or 16 patients with other liver diseases gave positive ADDC reactions. It was determined that of the 36 patients diagnosed with tienilic hepatotoxicity: 38% had both anti-LKM2 autoantibodies-and the tienilic acid-associated antibodies in their sera, 18% had only the anti-LKM2 autoantibodies, and 9% had only the tienilic acid-associated antibodies. Although the identity of the tienilic acid-altered antigen is not known, it most likely corresponds to a protein that has been covalently altered by reactive metabolites of tienilic acid. In this regard, it has recently been shown that when radioactively labeled tienilic acid is incubated with rat liver microsomes, it is metabolized by cvtochromes P450 into an unknown reactive intermediate that covalently binds to microsomal p r ~ t e i n . ~One ' possible candidate for the microsomal carrier protein of the reactive metabolite of tienilic acid might be cytochrome P450 8. If this were the case, then the tienilic acid-cytochrome P450 8 carrier conjugate might be the immunogen responsible for the induction of both the anti-LKM2 autoantibodies and the antibodies directed against the tienilic acid-induced NAD, as illustrated in Figure 1. Other liver proteins, however, might be the target of reactive metabolites of tienilic acid. Moreover, it is also possible that the neoantigens are not formed directly by reacting with metabolites of tienilic acid, as discussed earlier.

ETHANOL Alcoholic liver disease is a major cause of morbidity and mortality. In spite of extensive investigations, the mechanism by which alcohol produces hepatitis remains u n k n ~ w n . * ~The , * ~possibility that this disease may have an immune basis has been receiving considerable attention during the last 5 year^.^^-*^ An early indication that alcoholic liver disease might be mediated by an immunologic reaction was the finding that ethanol or acetaldehyde, the first metabolite of ethanol oxidation, induced

the transformation of lymphocytes from patients with acute alcoholic he pa ti ti^.^' These compounds had no effect on lymphocytes from individuals with normal liver, alcoholic fatty liver, or acute viral hepatitis. They did, however, stimulate lymphocytes from patients with chronic active hepatitis. It was additionally found that autologous liver homogenates produced the same degree of stimulation. Several more recent studies have shown that 38 to 74% of alcoholic hepatitis patients have antibodies in their sera that react with the surface of hepatocytes of rabbits that had been treated with a l ~ o h o l . ~No ~-~~ ethanol-related antibodies were found in the sera of normal individuals or in patients with other types of acute or chronic liver disease. It appears that acetaldehyde is responsible for the formation of neoantigens recognized by the patients' antibodies. The treatment of rabbits with 4-methylpyrazole (an inhibitor of alcohol dehydrogenase), or with disulfiram (an inhibitor of aldehyde dehydrogenase), decreased and increased, respectively, the amount of reaction of the patients' antibodies with the ~~ other primary alcohols rabbit h e p a t ~ c y t e s .Moreover, that are metabolized to aldehyde metabolites, such as methanol and propan-1-01, also sensitized rabbit hepatocytes so that they reacted with the patients' serum ant i b ~ d i e s . ~This ' finding indicates that it is the reactivity of the aldehyde functional group of acetaldehyde that is responsible for the formation of the liver neoantigens recognized by the patients' serum antibodies. Acetaldehyde has been shown to react in vitro with a variety of proteins, including albumin, plasma proteins, erythrocyte membrane proteins, hepatic microsoma1 proteins, hemoglobin, and tub~lin.'~."It forms both unstable and stable adducts. The unstable adducts appear to be Schiff-base derivatives of lysine residues because they can be converted to stable N-ethyl lysine adducts by treatment with sodium borohydride or sodium cyanoborohydride." Not as much is known about the structures of the stable adducts, except in the case of hemoglobin where they appear to be imidazolidinone derivatives of the N-terminal valine residues of both a and p chain^.^' Acetaldehyde-protein adducts (APAs), formed from the reaction of acetaldehyde with keyhole limpet hemocyanin, serum albumin, prothrombin, hemoglobin, or other proteins in the presence of sodium borohydride or sodium cyanoborohydride, have been used as test antigens for detecting antibodies in the sera of alcoholic patients"234 and in the sera of mices5 and rats3h treated chronically with alcohol. The serum antibodies were found to react with more than one of the APAs, indicating that the epitopes recognized by the antibodies were not carrier-protein specific, but instead corresponded to similar if not identical covalently bound acetaldehyde haptenic groups. In contrast to the assays that use hepatocytes from rabbits treated with ethanol as the test an tiger^,^^-^^ a problem with the APA assays is that they are not specific for patients with alcoholic liver disease. Patients with nonalcoholic liver disease, such as chronic active hepatitis, primary biliary cirrhosis, and viral hepatitis, also

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react with the APAs, although generally less frequently and with a lower titer of antibody than that of the alcoholic patients.j2 3 4 It appears that the synthetic APAs are not specific enough antigens to detect serum antibodies unique to patients with alcoholic liver disease. Moreover, there is no evidence that rats and mice that are fed ethanol chronically and develop serum antibodies that react with the APAs show any signs of hepatotoxicity.'s.'h The best antigens to use, not only for detecting patients sensitized to alcohol, but also for determining the role of the immune system in alcohol liver disease, should be those proteins in the liver that are specifically altered by alcohol. In this regard, it has been found that when radiolabeled ethanol was incubated with liver slices, the radiolabel bound irreversibly to liver p r ~ t e i n . ~The ' binding appeared to be mediated by acetaldehyde, at least in part, because the alcohol dehydrogenase inhibitor pyrazole decreased, whereas the aldehyde dehydrogenase inhibitor cyanamide increased the level of binding of the radiolabel. Other investigators, using high pressure liquid chromatography (HPLC), have detected an adduct in the acid hydrolysate of liver plasma membranes isolated from rats fed alcohol that had a similar retention time to ' ~ protein or a synthetic acetaldehyde-lysine a d d ~ c t . The proteins in the plasma membrane from which the lysineacetaldehyde adduct was derived are not yet known. More recently, a 37 kd neoantigen has been detected in the liver of rats fed alcohol chronically by immunoblotting liver subcellular fractions with anti-APA antibodie~.~'."The adduct was found in cytosol, but not in the microsomal or mitochondria1 fractions of the liver. An apparently identical 37 kd adduct is formed when primary cultures of rat hepatocytes are exposed to ethanol for several days.4' It appears that acetaldehyde is responsible for the formation of the 37 kd neoantigen, not only because the anti-APA antibodies recognize the protein, but also because the amount of the protein increases when the acetaldehyde dehydrogenase inhibitor cyanamide is added either to the diet of rats4" or the culture media of hepatocytes exposed to ethanol.41The authors do point out that it is still possible that the antiAPA antibodies might recognize instead a naturaloccurring N-ethylated lysine moiety of a 37 kd protein whose concentration is increased by ethanol. Although the identity of the 37 kd is not known, immunosorption studies rule out the possibility that it corresponds to alcohol dehydrogenase or aldehyde dehydrogena~e.~" In contrast to these findings, other investigators have detected a 52 kd neoantigen in the liver microsomes of alcohol-fed rats using anti-APA antibodies." The protein was isolated by immunoaffinity chromatography on a column of Sepharose-conjugated anti-APA immunoglobulin G (IgG) and was found to correspond to cytochrome P450IIE1, the microsomal enzyme that is induced by ethanol and oxidizes it to acetaldehyde. It has been suggested that the cytochrome P4501IEl adduct does not arise from the covalent interaction of acetaldehyde with this enzyme, but instead is formed from a reactive metabolite of ethanol that does not escape from cytochrome P4501IE1. This idea is based in part on the fact that stable acetaldehyde adducts were not detected

10, NUMBER 4, 1990

with other microsomal proteins. One possible reactive species that could be responsible for the covalent alteration of cytochrome P450IIE1 is the I-hydroxyethyl radical metabolite of ethanol, since this species has been spin-trapped from the reaction mixture of ethanol and liver of microsomes of rats.4' Although the 37 kd and 52 kd neoantigens react with the anti-APA antibodies, it remains to be determined whether these neoantigens are recognized by the antibodies in the sera of the alcoholic hepatitis patients. Immunoblotting liver tissue fractions from alcoholtreated animals with the patients' antibodies might be one way of answering this question. It may also lead to the detection of other alcohol-related liver neoantigens.

HALOTHANE HEPATITIS The elucidation of the mechanism of a rare but severe form of hepatitis caused by the inhalation anesthetic halothane (CF,CHC1Br)10~44~4s is of clinical interest, because halothane is still widely used in adults in the ~ . ~is' often an agent of United Kingdom and E ~ r o p e ~and choice for ~ h i l d r e n . ~Moreover, ~ - ~ ~ recent reports suggest that the structurally related inhalation anesthetic enflurane (CHF,0CF2CHFCI), which is widely used in adults in the United States, may also cause liver damage " ~ ~most important discovery by a similar p r o c e s ~ . ~The that led to the current understanding of halothane-induced hepatitis was the finding over 10 years ago that a high proportion of the patients with this toxicity contained specific lymphocytes and antibodies in their blood that were directed against undefined halothane-altered liver neoantigens."' For example, a leukocyte cell migration test suggested that 8 of 12 patients with unexplained fulminant hepatic failure after halothane had specific Tcell lymphocytes in their blood that recognized cellular determinants in a subfraction of liver homogenates from halothane-treated rabbits. Similar evidence for cellular immunity to a neoantigen formed during halothane anesthesia was also obtained in three of four patients by a direct lymphocyte cytotoxicity assay. Moreover, specific circulating IgG antibodies directed against halothane-altered tissue components were detected in the sera of several halothane hepatitis patients by either an indirect immunofluorescence procedure (7 of 1 1 patients) or a more sensitive and rapid enzyme-linked immunosorbent assay (ELISA; 16 of 24 patients). Further characterization of the halothane-induced neonantigens by immunoblotting with sera from several halothane hepatitis patients has revealed that the neoantigens correspond to at least five polypeptides fractions (100, 76, 59, 57, and 54 kd) that are expressed predominantly in the microsomal fraction of the l i ~ e r . ~ ' ~ ~ ~ More recently, the 76 kd has been calculated to have an apparent monomeric mass of 80 kd;" therefore in the future, it will be called the 80 kd neoantigen. It appears that similar neoantigens are formed in the livers of rabbits. ,9.51 and humanss4 treated with halothane, indicating that liver microsomes of animals and humans contain structurally related carrier proteins. Although patients can be sensitized against different neoantigens

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FIG. 2. Detection of neoantigens in liver microsomes of halothane-treated rats by immunoblotting with sera from three halothane hepatitis patients. Constituent polypeptides were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred electrophoretically to nitrocellulose, and reacted with patients' antibodies. Lane A is the immunoblot using liver microsomes from untreated rats; Lane B is the immunoblot derived from liver microsomes from halothane-treated rats. The antibodies in the sera of the patients react with the following halothane-induced neoantigens: patient l , 100 and 80 kd neoantigens; patient 2, 100, 80, 59, and 54 kd neoantigens; patient 3, 57 kd neoantigen. (Adapted from Pohl et al. lo6)

(Fig. 2), each neoantigen has covalently bound to it the same trifluoroacetyl (TFA) hapten, which is derived from the reactive metabolite, trifluoroacetyl chlorides2 (Fig. 3). This was initially indicated by the finding that the neoantigens in liver microsomes from halothanetreated rats, which were recognized by the patient's antibodies, also appeared to be recognized by a haptenspecific anti-TFA antibody5' (Fig. 4). It was conclusively established by three additional experiments that the TFA hapten is responsible for the generation of the halothane-carrier conjugate^.^^

Halothane

Enflurane

F H 1 I F-c-c-CI I I F Br F F H I I I F-C-0-C-C-CI I I /

- -

-

F 0 I II F-C-C-CI I F

F O I l l

F I

F-c-o-c-c-F I

F 0 I II F-C-C-P

P

p

-o

F I

F O I l l

I

I

F-c-o-c-c-P

I

or

I F

-

F-C-C-P

FIG. 3. Metabolic pathways for the formation of acylated liver proteins (P) following the administration of halothane, enflurane, or isoflurane. In each case, the acyl halide is produced by an oxidative dehalogenation reaction catalyzed by cytochromes P450. P: cellular proteins.

FIG. 4. Detection of trifluoroacetylated (TFA) proteins in liver microsomes of halothane-treated rats by immunoblotting with hapten-specific anti-TFA antibodies. Constituent polypeptides were resolved by sodium dodecyl sulfatepolyacrylamide gel electrophoresis, transferred electrophoretically to nitrocellulose, and reacted with hapten-specific antiTFA antibodies as previously d e s ~ r i b e dThe . ~ ~ hapten-specific anti-TFA antibodies reacted with several microsomal proteins. Purified TFA proteins of 100, 80, 63, 59, or 57 kDa have also been shown by an enzyme-linked immunosorbent assay procedure to react with antibodies in the sera of halothane hepatitis patients.53A TFA protein of 58 kDa, which is not resolved from the TFA-59 kd on this immunoblot, also reacts with antibodies in the sera of several of the halothane patients.53

First, it was found that when rats were treated with deuterium-labeled halothane (CF,CDCIBr) in place o f halothane, a smaller amount of the neoantigens was produced. This showed that the rate-determining step in the formation of the neoantigens was the cleavage of the C-H bond of halothane. Second, the neoantigens were formed in vitro only when halothane was incubated with liver microsomes aerobically and not anaerobically, confirming that the reactive metabolite was formed by an oxidative process. This ruled out the possibility that the neoantigens were formed by the reductive radical metabolite of halothane, CF,CHCI. Third, it was found that the removal of the TFA haptens from the rat liver microsomal proteins by treatment with 1 M piperidine abolished nearly all of the reaction of the patients' antibodies with the halothane-induced neoantigens. It has also been found that the oxidative metabolism of halothane to trifluoroacetyl chloride by microsomal cytochromes P450 occurs predominantly in the liver;55 this probably explains the organ specificity of halothane toxicity. It has been concluded that the epitopes recognized by the patients' antibodies are not solely the bound TFA

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SEMINARS IN LIVER DISEASE-VOLUME

hapten, but instead the TFA hapten and portions of the specific carrier proteins."'.' This follows from the findings that patients' sera differ in their patterns of polypeptide neoantigen recognition and that inhibition of binding of the patients' antibodies to the TFA-proteins by the hapten derivative N-epsilon-TFA-lysine (TFA-lysine) is weak, even at very high concentrations.

Possible Metabolic and immunologic Basis for Hepatitis Produced by Other Inhalation Anesthetics Enflurane (CHF20CF2CHFCI) and isoflurane (CHF20CHC1CF,), two other polyhalogenated volatile anesthetics, were developed because of their decreased rates of metabolism and hence decreased potential for toxicity. However, case reports of enflurane hepatitis and unexplained hepatic dysfunction following isoflurane anesthesia have appeared, although much less frequently than those describing halothane he pa ti ti^.^' The suggestion has also been made that a cross-sensitization may exist between prior anesthesia with halothane and the subsequent production of hepatic necrosis after enflurane exposure. However, no mechanistic explanation for these possibilities had been proposed until recently. Enflurane and isoflurane, like halothane, are expected to be oxidatively metabolized by cytochromes P450 into reactive products that can acylate proteins"' (Fig. 3). In fact, in the case of isoflurane, TFA adducts should be formed. The use of anti-TFA antibodies in immunoblotting procedures revealed that enflurane and isoflurane also form acylated protein adducts in the livers of rats." The relative order of immunoreactive protein adducts formed was halothane > enflurane > isoflurane. This correlates directly with the relative extent of metabolism of these agents. Even more significantly, when immunoblotting was done with sera of six patients with halothane hepatitis instead of anti-TFA antibodies, crossreaction occurred with proteins in liver microsomes of the enflurane-treated rats."' The neoantigens produced in the liver microsomes from the rats treated with isoflurane, however, were beyond the limits of detection of the immunoblotting procedure. The results of these studies can explain the apparent cross-sensitization between halothane and enflurane anesthesia and how enflurane or possibly isoflurane may also cause hepatotoxicity by immune-mediated processes.

Characterization of the 59 kd Liver Microsomal TFA-Carrier Conjugate Recently, several of the TFA-carrier conjugates have been characterized. The basic approach has been to determine whether the amino acid sequences of their N-terminals or internal peptides correspond to the sequences of known proteins. The TFA-59 kd protein was purified from liver microsomes of halothane-treated rats by first isolating the TFA-proteins from liver microsomes using an immunoaffinity column of anti-TFA IgG and then purifying the TFA-59 kd protein from this mixture by anion exchange HPLC. Based on its apparent monomeric molecular mass, NH2-terminal amino acid

10, NUMBER 4, 1990

sequence, catalytic activity, and other physical properties, the protein was identified as a microsomal c&boxyle s t e r a ~ e , ~which ' appears to be identical to form E l ." This isozyme apparently corresponds to form ES-81 ES-10.s9 At the time of the purification of the TFA-59 protein, there was no complete sequence information available for any mammalian liver carboxylesterase. Thus, a liver carboxylesterase cDNA was cloned from a rat liver lambda gtl 1 expression library by the use of a polyclonal antibody raised against the TFA-59 kd protein as a screening reagent.'() he cDNA clone encoded for a carboxylesterase that showed overall identity of 84% with several peptides derived from the 59 kd protein. It appeared therefore that an isoform of the 59 kd protein had been cloned. Other investigators have apparently cloned the same rat liver ~arboxylesterase.~' It is not yet known if any of the liver microsomal carboxvlesterase isoforms biochemically characterized to date correspond to the cloned cDNA. The carboxylesterases are a family of enzymes based on their substrate specificity and electrophoretic and immunologic proper tie^."-'^ Although the genes of these enzymes have not yet been cloned, genetic studies in animalssyas well as Southern blot analysis of genomic DNAho suggest that the enzymes comprise a multigene family. These enzymes may have several functions in the body. They have been shown to hydrolyze not only ester, thioester, phosphoester, and amide bonds of xenobiotics, but also endogenous lipids, such as fatty acid esters of carnitine and coenzyme A (CoA), acylglycerols, lysophospholipids, and phospholipid^.'^ " One of the carboxylesterases, also known as egasyn, forms a complex with p-glucuronidase. This interaction is thought to result in the specific localization of p-glucuronidase within the lumen of the endoplasmic reticulum (ER)." The 59 kd carboxylesterase, however, does not have this activity.'" Whether any of the carboxylesterases have the function of anchoring other proteins in the lumen of the ER is not known. The potential biologic importance of the carboxylesterase is also indicated by the finding that nearly all of the tissues studied to date contain carboxylesterase activities."."

Characterization of the Other Liver Microsomal TFA-Carrier Conjugates Another approach has been used to purify the TFAneoantigens. It was recently found that the 100, 80, 59, and 57 kd TFA neoantigens could be selectively extracted from liver microsomes of halothane-treated rats by 0.1% deoxycholate detergent treatment. The individual proteins were purified from this mixture by a combination of DEAE Sepharose anion exchange chromatography and HPLC.53 During the purification of the proteins, two additional TFA proteins were discovered. One has an apparent monomeric molecular mass of 58 kd, whereas the other is 63 kd." The TFA-57 kd protein corresponds to protein disulfide isomerase7' and appears to have multiple physiologic functions. Not only does it catalyze the isomerization of both the intramolecular and intermolecular disulfide bonds of proteins yielding apparently native

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3 10

structures, but it also appears to be the P-subunit of proline-4-hydroxylase, the major non-nuclear binding protein of thyroid hormone (3,3',5-triodo-L-thyronine),and the glycosylation site binding protein component of oligosaccharide tran~ferase.~' It is also concentrated within the lumen of the ER.73 The TFA-100 kd protein has been identified as E R P ~ which ~ , ~ is~ believed to be identical to a 94 kd glucose-regulated protein (grp94), and endoplasmin.74-77Although the physiologic function of this protein is not known, it appears to have at least two activities. It is a calcium-binding protein and may have a role in cellular calcium h ~ m e o s t a s i s . ~ 'The . ~ ~ protein also binds malfolded proteins. It has been speculated that this activity may have a role in preventing the secretion of malfolded proteins, in disposing of malfolded protein aggregates, or in renaturing malfolded protein^."^^^ Like the 59 and 57 kd proteins, the 100 kd protein is also concentrated within the lumen of the ER. Several studies have indicated that the localization of the 100 and 57 kd proteins in the lumen of the ER is due to the presence of a KDEL sequence at their C-terminals." This structural motif, however, does not appear to be the only signal that retains proteins in this subcellular ~ompartment;~' the C-terminal of the carboxylesterase encoded by the rat liver cDNA has the sequence TEHT.M'.h' Amino acid sequence analyses of the TFA-63 protein and a partial cDNA clone show that the TFA-63 kd neoantigen is ~alregulin.~"he protein is localized in the ER and is thought to have a role in calcium homeostasis, since it is a major site of calcium storage in the liver.80 The identities of the TFA-80 kd and TFA-58kd proteins are not conclusively known at this time.

Enzyme-linked lmmunosorbent Assays Using the Liver Microsomal TFA-Carrier Conjugates An assay for the detection of the antibodies directed against the TFA neoantigens has potential clinical importance for several reasons. First, the presence of the antibodies in the sera of patients previously exposed to halothane would indicate prior sensitization and would identify patients at increased risk for developing a hypersensitive reaction on further exposure to halothane. Second, these same antibodies have been shown to react with liver microsomal neoantigens produced by the structurally related inhalation anesthetic enflurane. This suggests the potential for a cross-sensitization reaction ~ ~ . ~ ~the assay can be after enflurane e x p o s ~ r e . Third, used to monitor sensitization to halothane in animal model studies of the immunopathologic mechanism of halothane he pa ti ti^.^' 8 3 Two types of immunochemical assays have been reported for the routine detection of the patients' antibodies. The first method is immunoblotting, using microsoma1 proteins from halothane-treated rabbits or rats as the test antigen. By this method, 42 of 68 (62%) patients with halothane hepatitis have been found to test positive for the halothane-induced antibodies." Although this approach provides important information concerning the apparent molecular mass of the neoantigens reacting

with the patients' antibodies, it is laborious and time consuming. It also has the potential of being inherently less sensitive than other methods, because it involves the protein denaturing conditions of sodium dodecyl sulfatepolyacrylamide gel electrophoresis. This could lead to a decreased level of response if a patient's antibodies were directed against, at least in part, conformational epitopes of the TFA ne~antigens.'~ The second immunochemical assay that has been employed for the detection of antibodies in the sera of halothane hepatitis patients is based on the more rapid, facile, and potentially more sensitive ELISA methodology. In this test, antigen is applied directly to the wells of a microtiter plate. One ELISA assay has employed the TFA hapten as test antigen in the form of TFA-rabbit serum albumin (TFA-RSA). Positive responses of halothane hepatitis patients using this antigen have been two of six (33%) patients," 16 of 24 (67%) patients,'" five of six patients (83%),X7and 12 of 40 (30%) patient^.'^ Another procedure, which appears to be more specific for detecting patients sensitized to h a l ~ t h a n e , ~utilizes ' microsomes from halothane-treated rabbits as test antigen. Employing this approach, antibodies have been detected in the sera of 16 of 24 (67%)" and 28 of 39 (72%)") of the patients with halothane hepatitis. Recent results indicate that the specificity and sensitivity of the ELISA procedure can be increased even further by utilizing purified rat liver microsomal TFAneoantigens as test antigens in place of the mixture of microsomal proteins. This should allow a higher concentration of the epitopes of each antigen to come in contact with the patient's antibodies. In a preliminary study, 79% of the halothane hepatitis patients tested positive for the halothane-induced antibodies, when the test antigens were the TFA-100, TFA-80, and TFA-57 kd neoantigens." It is anticipated that this value could be increased to over 90% if all of the TFA-neoantigens were included as test antigens, since some patients might not be sensitized to the TFA-57, TFA-80, or TFA-100 kd neoantigen.

POSSIBLE PATHWAYS FOR LIVER NEOANTIGENS TO INTERACT WITH THE IMMUNE SYSTEM AND LEAD TO LIVER IMMUNOPATHOLOGY One of the questions that has to be answered to explain the immunologic basis of drug-induced hepatotoxicity is how the immune system comes in contact with neoantigens that are formed intracellularly. There are several pathways that can be postulated to account for these interactions (Fig. 5). In Pathway 5A, a neoantigen formed intracellularly becomes exposed to the extracellular space when some hepatocytes are damaged by the intrinsic toxic effects of the drug. In the extracellular space, they come in contact with immunocompetent cells and induce a primary immune response. On further exposure to the drug, the release of the neoantigens to the extracellular space results in the formation of immune complexes at this site, which may activate complement and result in a localized inflammatory response (Fig. 6C).%Alrnatively, it may be possible for immu-

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Reactive Metabolite /

FIG. 5. Possible pathways for the immune system to come in contact with neoantigens produced by reactive metabolites of drugs. Pathway A involves

nocompetent cells to interact with the neoantigen without invoking cell death, if it is expressed on the exterior of the plasma membrane. This may occur if the reactive metabolite is long-lived enough to diffuse to the plasma membrane and covalently bind to macromolecules at this site (Fig. 5B). It is believed. however, that a more predominant pathway for drug-induced neoantigens to be expressed on the cell surface involves their initial intracellular formation, followed by translocation to the plasma membrane (Fig. 5C). In the case of several of the TFA-neoantigens associated with halothane hepatitis, there is a reasonable explanation for this trafficking. As discussed earlier in this review, current evidence indicates that the 100, 59, and 57 kd proteins are concentrated within the lumen of the ER. Most proteins that are secreted or become components of the plasma membrane, traffic to

this region of the ER.73Moreover, a recent study indicates that proteins normally residing in the lumen of the ER can be secreted when a cell is perturbed, such as by an increase in intracellular calcium.77Alternatively, the trafficking of a protein might be altered if it is covalently modified by a reactive drug metabolite. It may also be possible for neoantigens or peptides derived from them to be transferred to the plasma membrane by antigen-processing pathways involving the major histocompatibility complex (MHC) class I or class I1 molecules." Although hepatocytes normally express only low levels of these immunologically important molecules, under certain conditions their levels may become elevated and hepatocytes may act as antigen-presenting cells as well as cellular targets of the immune system,'? '4 Indeed. the abnormal expression of these molecules is thought to play a major role in the development of chronic active hepatitis," primary biliary c i r r h o ~ i s , ~ ~ and viral he pa ti ti^.'^ There are several potential ways hepatocytes might be damaged once the neoantigen is expressed on the surface of the plasma membrane. These include effector pathways involving complement or ADCC (Fig. 6A).' Alternatively, if the neoantigen or peptides derived from it are associated on the cell surface with class I or class 11 MHC antigens, then a specific cellular cytotoxicity might occur (Fig. 6B).' -

FUTURE STUDIES

FIG. 6. Possible immunologic pathways of drug-induced hepatitis. Pathway A involves reactions at the plasma mem-

brane that are mediated by antibodies. The effectors for this pathway could be either complement (C) or cells that have Fc receptors (antibody-dependentcellular toxicity [ADCC]).Pathway B is a specific cellular cytotoxicity mechanism. Pathway C occurs by a localized extracellular inflammatory response mediated by immune complexes and complement.

The initial objectives of future studies should be the purification and identification of hapten-carrier conjugates. Although in the past this seemed an insurmountable task, this is no longer the case, as shown by the studies with halothane. Recent major progress in the understanding of how drugs are metabolized into reactive metabolites, in the development of various immunochemical procedures, particularly immunoblotting and ELISA procedures, and in the molecular cloning of cellular proteins makes such studies possible. Once the hapten-carrier conjugates are purified, they could have sev-

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the release of neoantigens from dead cells. Pathway 6 represents the formation of neoantigens at the site of the plasma membrane. This could occur by reactive metabolites diffusing to this region. Pathway C involves trafficking of intracellular neoantigens to the plasma membrane either by normal trafficking routes or those that are mediated by MHC class I or II antigens.

era1 important applications. First, they could be used to develop specific and sensitive ELISA procedures for identifying patients sensitized to the drug. Second, by knowing the structure of the hapten, potentially safer derivatives of the drug could be developed, which would not be metabolized into reactive metabolites. Third, the hapten-carrier conjugates could be used in developing an animal model of the drug-induced hepatitis. This could be approached by immunizing animals with the haptencarrier conjugate. Once a humoral and cellular immune response is obtained, then the animal could be challenged with the drug to express the neoantigens in hepatocytes. If toxicity is obtained, then the immunopathologic basis of the toxicity could be studied in detail. Similar approaches are currently being used to determine the mechanism of most autoimmune diseases. Fourth, if the purified carrier proteins can be identified, it should be possible to determine whether their physiologic activities are altered when they are covalently modified by a reactive metabolite. Altered activities may have a role in the hepatoxicity caused by the drug.

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